Interleukin-18 acts as an angiogenesis and tumor suppressor.

Interleukin-18 (IL-18), also called interferon-gamma (IFN-gamma)-inducing factor, has recently been characterized as a potent IFN-gamma-inducing cytokine. We now report that IL-18 is a novel antiangiogenic and antitumor cytokine. In vitro, IL-18 specifically inhibits fibroblast growth factor-2-stimulated proliferation of capillary endothelial cells. In vivo, IL-18 is sufficiently potent to suppress the fibroblast growth factor-induced corneal neovascularization by systemic administration in mice. This cytokine also inhibits embryonic angiogenesis in the chick chorioallantoic membrane assay. Systemic and intralesional administrations of IL-18 produce a significant suppression of the growth of murine T241 fibrosarcoma in syngeneic C57Bl6/J and immunodeficient SCID mice. The antitumor effect appears to be potent because an average of >75% inhibition of primary tumor growth was observed at a dose of 50 microg/kg/day. In cell culture, murine T241 fibrosarcoma cells are insensitive to recombinant IL-18 at concentrations that significantly inhibit endothelial cell proliferation. Immunohistochemical studies of tumor tissues reveal hypovascularization of the IL-18-treated tumors. These results suggest that IL-18 may participate in the regulation of a switch of tumor angiogenesis.-Cao, R., Farnebo, J., Kurimoto, M., Cao, Y. Interleukin-18 acts as an angiogenesis and tumor suppressor.     

Application of plasmid DNA encoding IL-18 diminishes development of herpetic stromal keratitis by antiangiogenic effects

HSV-1 infection of the eye can cause a blinding immunoinflammatory stromal keratitis (SK) lesion. Using the mouse model, we have demonstrated that angiogenesis is an essential step in lesion pathogenesis because its inhibition results in diminished severity. The molecules involved in causing corneal angiogenesis are multiple and include the vascular endothelial growth factor (VEGF) family of proteins. In this report we show that application of plasmid DNA encoding IL-18 to the cornea of mice before HSV-1 ocular infection resulted in reduced angiogenesis and diminished SK immunoinflammatory lesions. The antiangiogenic effects of IL-18 treatment appeared to be mediated by inhibition of VEGF production in the cornea. We also showed that IL-18 controlled VEGF expression in vitro and also decreased CpG oligodeoxynucleotide induced VEGF-dependent neovascularization. In addition the administration of IL-18-binding protein, an IL-18 antagonist, into the inflammatory eye resulted in elevated angiogenesis and increased VEGF expression. Our results indicate that IL-18 is an important endogenous negative regulator of HSV-induced angiogenesis resulting in reduced SK lesion severity. Our results could mean that IL-18 administration may represent a useful approach to manage unwanted angiogenesis.     

Antiangiogenic and antitumor activities of IL-27

IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.     

Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, _D(KLAKLAK)₂, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the αvβ3 and αvβ5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C–_D(KLAKLAK)₂ chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.     

Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model

Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase high-performance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC-4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases.     

Broad Spectrum Antiangiogenic Treatment for Ocular Neovascular Diseases

Pathological neovascularization is a hallmark of late stage neovascular (wet) age-related macular degeneration (AMD) and the leading cause of blindness in people over the age of 50 in the western world. The treatments focus on suppression of choroidal neovascularization (CNV), while current approved therapies are limited to inhibiting vascular endothelial growth factor (VEGF) exclusively. However, this treatment does not address the underlying cause of AMD, and the loss of VEGF''s neuroprotective can be a potential side effect. Therapy which targets the key processes in AMD, the pathological neovascularization, vessel leakage and inflammation could bring a major shift in the approach to disease treatment and prevention. In this study we have demonstrated the efficacy of such broad spectrum antiangiogenic therapy on mouse model of AMD.

Methods and Findings

Lodamin, a polymeric formulation of TNP-470, is a potent broad-spectrum antiangiogenic drug. Lodamin significantly reduced key processes involved in AMD progression as demonstrated in mice and rats. Its suppressive effects on angiogenesis, vascular leakage and inflammation were studied in a wide array of assays including; a Matrigel, delayed-type hypersensitivity (DTH), Miles assay, laser-induced CNV and corneal micropocket assay. Lodamin significantly suppressed the secretion of various pro-inflammatory cytokines in the CNV lesion including monocyte chemotactic protein-1 (MCP-1/Ccl2). Importantly, Lodamin was found to regress established CNV lesions, unlike soluble fms-like tyrosine kinase-1 (sFlk-1). The drug was found to be safe in mice and have little toxicity as demonstrated by electroretinography (ERG) assessing retinal and by histology.

Conclusions

Lodamin, a polymer formulation of TNP-470, was identified as a first in its class, broad-spectrum antiangiogenic drug that can be administered orally or locally to treat corneal and retinal neovascularization. Several unique properties make Lodamin especially beneficial for ophthalmic use. Our results support the concept that broad spectrum antiangiogenic drugs are promising agents for AMD treatment and prevention.     

IFN-gamma and IL-2 are protective in the skin but pathologic in the corneas of HSV-1-infected mice.

HSV type 1 (HSV-1) infection of the mouse cornea results in a tissue-destructive inflammatory reaction in the cornea, but little or no disease in the skin surrounding the eye. Depleting T lymphocytes from mice before HSV-1 corneal infection prevents the corneal inflammation but severely exacerbates the periocular skin lesions. Studies described in this communication investigated the role of T cell cytokines in the corneal and periocular skin disease induced by HSV-1 corneal infection. Mice received weekly i.p. injections of rat mAb specific for IL-2, IL-4, or IFN-gamma beginning 1 day before (day -1) or 6 days after (day +6) corneal infection with the RE strain of HSV-1. The severity of corneal inflammation and the area of periocular skin involvement were measured. Treatment with anti-IFN-gamma or anti-IL-2 significantly reduced the incidence and severity of corneal inflammation. Treatment was equally effective when initiated on day -1 (before T cell activation) or day +6 (after T cell activation but before the initiation of corneal inflammation). Treatment with anti-IL-4 had no effect. The histologic features of corneal inflammation in mock-treated mice included neovascularization, corneal edema, and cellular infiltration. Corneas of anti-IL-2-treated mice that developed inflammation had similar but less severe histologic features. Corneas of anti-IFN-gamma-treated mice that developed inflammation had neovascularization and edema but minimal cellular infiltration. Treatment with anti-IFN-gamma or anti-IL-2 significantly exacerbated periocular skin lesions when initiated at day -1, but not when initiated at day +6. Anti-IL-4 treatment had no effect on skin lesions. Treatment with either anti-IFN-gamma or anti-IL-2, when initiated at day -1, significantly inhibited the delayed-type hypersensitivity response to HSV Ag, but when treatment was begun at day +6 only anti-IFN-gamma significantly inhibited the delayed-type hypersensitivity response. Our findings suggest that IFN-gamma and IL-2 are important elements in both an immunopathologic T-lymphocyte response to HSV-1 Ag in the cornea and a protective T lymphocyte response in the skin.     

Recombinant fragment 44–77 of the pigment epithelium-derived factor prevents the development of the pathological cornea neovascularization

The pigment epithelium-derived factor (PEDF), a secreted 50 kDa glycoprotein, is one of the most potent endogenous inhibitors of angiogenesis. The fragment 44–77 of PEDF possesses the antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of diseases of visual organs accompanied by pathological neovascularization. An effective biotechnological method for the large-scale production of the PEDF (44–77) fragment as part of the fusion protein with the SspDnaB intein has been developed. The hybrid protein was produced in Escherichia coli bacterial cells in the form of inclusion bodies, solubilized, and subjected to autocatalytic cleavage with the release of the PEDF (44–77) fragment (reaction yield 77%). The target peptide was separated from the intein by tangential ultrafiltration. The final purification of PEDF (44–77) was performed by reversed-phase HPLC. The yield of the target peptide (purity 99%) was 65 mg per 1 l of culture. The antiangiogenic activity of the peptide was confirmed in vitro using mouse endothelial cells SVEC-4-10. It was found that the preparation at a concentration of 1 nM suppresses the proliferation of endothelial cells by 53% and inhibits the formation of tube-like structures by endothelial cells. The ability of the recombinant PEDF (44–77) to block the initial stages of angiogenesis was shown using an experimental model of rabbit corneal neovascularization.     

Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.     

Robo 4 Counteracts Angiogenesis in Herpetic Stromal Keratitis

The cornea is a complex tissue that must preserve its transparency to maintain optimal vision. However, in some circumstances, damage to the eye can result in neovascularization that impairs vision. This outcome can occur when herpes simplex virus type 1 (HSV-1) causes the immunoinflammatory lesion stromal keratitis (SK). Potentially useful measures to control the severity of SK are to target angiogenesis which with herpetic SK invariably involves VEGF. One such way to control angiogenesis involves the endothelial receptor Robo4 (R4), which upon interaction with another protein activates an antiangiogenic pathway that counteracts VEGF downstream signaling. In this study we show that mice unable to produce R4 because of gene knockout developed significantly higher angiogenesis after HSV-1 ocular infection than did infected wild type (WT) controls. Moreover, providing additional soluble R4 (sR4) protein by subconjunctival administration to R4 KO HSV-1 infected mice substantially rescued the WT phenotype. Finally, administration of sR4 to WT HSV-1 infected mice diminished the extent of corneal angiogenesis compared to WT control animals. Our results indicate that sR4 could represent a useful therapeutic tool to counteract corneal angiogenesis and help control the severity of SK.     

Combined effects of IL-12 and IL-18 on the induction of collagen-induced arthritis

IL-18 expression has recently been detected in rheumatoid arthritis (RA) synovial membrane. We investigated the mechanisms by which IL-18-induced collagen-induced arthritis in DBA/1 mice primed intradermally with type II bovine collagen in IFA and boosted i.p. 21 days later with CII in saline. Mice were injected i.p. with rIL-12, rIL-18, or both (100 ng) during days -1 to 4 and again on days 20-24. Control mice received PBS. Mice treated with IL-12 or IL-18 alone developed significantly higher incidence and more severe disease compared with controls. These were elevated further by combination treatment with IL-12 and IL-18. The cytokine treatments led to markedly enhanced synovial hyperplasia, cellular infiltration, and cartilage erosion compared with controls. Cytokine-treated mice produced significantly more IFN-gamma, TNF-alpha, and IL-6 than the controls. Interestingly, IL-18-treated mice produced more TNF-alpha and IL-6, but less IFN-gamma, compared with mice treated with IL-12. Furthermore, splenic macrophages from DBA/1 mice cultured in vitro with IL-18, but not IL-12, produced substantial amounts of TNF-alpha. Mice treated with IL-18 or IL-18 plus IL-12 produced markedly more IgG1 and IgG2a anti-collagen Ab compared with controls, whereas IL-12 treatment only led to an enhanced IgG2a response. Together these results demonstrate that IL-18 can promote collagen-induced inflammatory arthritis through mechanisms that may be distinct from those induced by IL-12.     

Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.     

On the mechanism of thrombin-induced angiogenesis: involvement of alphavbeta3-integrin

Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of alphavbeta3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with alphavbeta3 as demonstrated by direct binding of alphavbeta3 protein to immobilized thrombin. This interaction of thrombin with alphavbeta3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to alphavbeta3 or a specific peptide antagonist to alphavbeta3 can abolish all these alphavbeta3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to alphavbeta3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi.     

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.     

Recombinant fragment of pigment epithelium-derived factor (44-77) prevents pathological corneal neovascularization

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.     

Complete regression of established spontaneous mammary carcinoma and the therapeutic prevention of genetically programmed neoplastic transition by IL-12/pulse IL-2: induction of local T cell infiltration, Fas/Fas ligand gene expression, and mammary epithelial apoptosis

Using a novel transgenic mouse model of spontaneous mammary carcinoma, we show here that the IL-12/pulse IL-2 combination can induce rapid and complete regression of well-established autochthonous tumor in a setting where the host immune system has been conditioned by the full dynamic process of neoplastic progression and tumorigenesis. Further, this regimen inhibits neovascularization of established mammary tumors, and does so in conjunction with potent local induction of genes encoding the IFN-gamma- and TNF-alpha-inducible antiangiogenic chemokines IFN-inducible protein 10 and monokine induced by IFN-gamma. In contrast to untreated juvenile C3(1)TAg mice in which histologically normal mammary epithelium predictably undergoes progressive hyperplasia, atypical changes, and ultimately transition to overt carcinoma, the current studies also demonstrate a unique preventative therapeutic role for IL-12/pulse IL-2. In juvenile mice, early administration of IL-12/pulse IL-2 markedly limits the expected genetically programmed neoplastic transition within the mammary epithelium and does so in conjunction with enhancement of constitutive Fas and pronounced induction of local Fas ligand gene expression, T cell infiltration, and induction of apoptosis within the mammary epithelium. These events occur in the absence of a durable Ag-specific memory response. Thus, this novel model system demonstrates that the potent therapeutic activity of the IL-12/pulse IL-2 combination rapidly engages potent apoptotic and antiangiogenic mechanisms that remain active during the delivery of IL-12/pulse IL-2. The results also demonstrate that these mechanisms are active against established tumor as well as developing preneoplastic lesions.     

Lactadherin promotes VEGF-dependent neovascularization

Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.     

Systemic soluble Tie2 expression inhibits and regresses corneal neovascularization

This study was designed to determine if soluble Tie2 (sTie2) expression inhibits and regresses corneal neovascularization, and if VEGF contributes to its effect. The corneas of BALB/c mice were scraped and the mice were injected with either an adenovirus expressing soluble Tie2 (Ad.sTie2) or an empty adenoviral vector. When injected at the inhibition timepoint (one day prior to corneal injury), the mean percentage of neovascularized corneal area two weeks later in Ad.sTie2-treated mice vs. controls was 56.37+/-9.15% vs. 85.79+/-3.55% (p=0.04). At the regression timepoint (4 weeks after corneal scrape), the mean area of corneal neovascularization in Ad.sTie2-treated mice was 42.89+/-4.74% vs. 75.01+/-3.22% in the control group (p=0.007). VEGF expression was significantly higher in Ad.sTie2-treated mice at the inhibition timepoint and there was no significant difference at the regression timepoint. These findings suggest that sTie2 inhibits and regresses corneal neovascularization in a VEGF-independent manner.     

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.     

Protective roles of the fractalkine/CX3CL1-CX3CR1 interactions in alkali-induced corneal neovascularization through enhanced antiangiogenic factor expression

Macrophages accumulate during the course of corneal neovascularization, but its mechanisms and roles still remain elusive. To address these points, we herein examined corneal neovascularization after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is normally expressed by macrophages. After alkali injury, the mRNA expression of CX3CR1 was augmented along with accumulation of F4/80-positive macrophages and Gr-1-positive neutrophils in the corneas. Compared with wild-type mice, CX3CR1-deficient mice exhibited enhanced corneal neovascularization 2 wk after injury, as evidenced by enlarged CD31-positive areas. Concomitantly, the accumulation of F4/80-positive macrophages, but not Gr-1-positive neutrophils, was markedly attenuated in CX3CR1-deficient mice compared with wild-type mice. The intraocular mRNA expression of vascular endothelial growth factor (VEGF) was enhanced to similar extents in wild-type and CX3CR1-deifient mice after the injury. However, the mRNA expression of antiangiogenic factors, thrombospondin (TSP) 1, TSP-2, and a disintegrin and metalloprotease with thrombospondin (ADAMTS) 1, was enhanced to a greater extent in wild-type than CX3CR1-deificient mice. A double-color immunofluorescence analysis demonstrated that F4/80-positive cells also expressed CX3CR1 and ADAMTS-1 and that TSP-1 and ADAMTS-1 were detected in CX3CR1-positive cells. CX3CL1 enhanced TSP-1 and ADAMTS-1, but not VEGF, expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 wk, along with enhanced intraocular expression of TSP-1 and ADAMTS-1 but not VEGF. Thus, these observations indicate that accumulation of CX3CR1-positive macrophages intraocularly can dampen alkali-induced corneal neovascularization by producing antiangiogenic factors such as TSP-1 and ADAMTS-1 and suggest the potential therapeutic efficacy of using CX3CL1 against alkali-induced corneal neovascularization.